One attractive, efficacious strategy for reducing both the pain and morbidity associated with sickle cell disease (SCD) would be to induce fetal gamma-globin synthesis in adult erythroid cells. Such a strategy could be executed, in theory, either by forcing fetal gene-specific transcriptional activators to be inappropriately activated during adult erythropoiesis or by inhibiting the activity of adult stage fetal globin gene repressors. Using a combination of molecular genetics and biochemistry, we recently identified a potential definitive stage gamma-globin gene repressor (which we named DRED, for direct repeat erythroid-definitive). We cloned the DNA binding subunits of the repressor by purifying them from adult murine erythroid tissue culture cells, and the summary of our current evidence suggests that the large DRED repressor complex binds to direct repeat (DR1) sites in the epsilon- and gamma-globin gene promoters using two nuclear orphan receptors, TR2 and TR4, as the molecular scaffold upon which the larger DRED complex is assembled. Since TR2/TR4 heterodimers have been shown to repress other cellular genes, DRED could constitute an excellent target for therapeutic intervention in the treatment of SCD. Here we experimentally address five questions that will either confirm or refute the hypothesis that DRED might be an appropriate target for therapeutic intervention in the treatment of SCD and/or Cooley's anemia (beta-thalassemia). First, can we provide further evidence, using modified transgenic human beta-globinYACs, that the DR1 element in the gamma-globin gene promoter is the direct target of DRED repression? Second, can we provide additional biochemical evidence that the TR2/TR4 heterodimer is the basis for that repression? Third, will tissue-specific gain of function experiments (forced transgenic expression of TR2 and TR4 in erythroid cells) lead to precocious silencing of the endogenous murine or transgenic human embryonic/ fetal globin genes? Fourth, will conditional, erythroid tissue-specific loss of function of TR2 and TR4 (by either germ line inactivation or dominant negative repression) lead to ectopic synthesis of embryonic/fetal globin genes in definitive erythroid cells? Fifth, what are the components, other than TR2 and TR4, that constitute the 0.5 MDa DRED repression complex.